Endometriosis causes pain and/or infertility in 2 - 8% of women in the U.S., with an annual cost of more than $20 billion. Despite the clinical and financial impact of endometriosis, its pathogenesis and pathophysiology remain poorly understood and treatment options remain limited. We and others have demonstrated abnormal expression of genes regulated by progesterone (P) in eutopic endometrium of women with endometriosis, suggesting resistance to P. Given that P inhibits endometrial proliferation, sets the stage for apoptotic cell death at menstruation, and induces receptivity to embryo implantation, P resistance could contribute to the pathogenesis and pathophysiology of endometriosis by facilitating proliferation and survival of endometrial implants and by inhibiting embryo implantation. Current knowledge gaps include the amount and duration of P action required for normal endometrial function, the extent to which P requirements may be altered in women with endometriosis, and the functional consequences of P resistance. We have begun to define P action and requirements in unaffected women using a novel in vivo model of the human menstrual cycle, in which circulating P concentrations are defined experimentally. Preliminary data in fertile subjects validates our approach, defines the approximate minimum threshold serum P concentrations required for normal structural (~2-3 ng/mL) and functional (~5-10 ng/mL) endometrial differentiation, and identifies mRNA species that can serve as dose-sensitive markers of P action. Base on preliminary data and existing literature, we hypothesize: (1) that women with endometriosis exhibit abnormal endometrial structural and/or functional development at P concentrations that achieve normal differentiation in fertile controls, (2) that progesterone resistance can be overcome by supra-physiological P concentrations;and (3) that abnormal patterns of endometrial epithelial gene expression in affected women result from abnormal paracrine signaling in P-resistant stroma. To test the first two hypotheses, we will use our established in vivo model to directly compare P requirements for normal endometrial differentiation in fertile women to those in infertile women with and without endometriosis. The effects of P on endometrial structure will be assessed histologically and the functional effects of P will be assessed by qRT-PCR and microarray analysis of isolated epithelial and stromal cell fractions and immunostaining of tissue sections. To test the third hypothesis, we will combine normal epithelial cells with stromal cells from women with or without endometriosis as a xenograft in a murine host. Treatments with estrogen and varying doses of P will permit assessment of the stromal contribution to abnormal endometrial miRNA, mRNA, and protein expression. These studies will assess directly endometrial P resistance, the effect of stroma on abnormal endometrial function, provide dose-specific markers of P action, and establish P requirements in fertile and infertile women with and without endometriosis. These human data will provide a solid foundation for the development of novel diagnostic and therapeutic strategies for endometriosis. PUBLIC HEALTH RELEVANCE: Endometriosis, a cause of pelvic pain, infertility, and pelvic and abdominal organ damage, affects about 4% of reproductive age women in the United States, with an estimated cost of $22 billion. The proposed research will produce a better understanding of the role of progesterone in endometriosis, identification of critically needed biomarkers, as well as provide a better understanding of cellular and molecular mechanisms of disease. The information gained will allow development of better diagnostic tests and therapy.